Inflammatory signaling by macrophages in response to pathogens or tissue injury is the key determinant of the pathology and pathogenesis of infectious and non- infectious disease. Signaling is initiated by Toll-like receptors (TLRs) and results in complex gene expression programs. The transcription factor NF?B controls the inflammatory portion of the program, and understanding its regulation in response to TLR signaling is the goal of this project. During the previous funding period, we have used combined experimental / computational modeling approach to gain a predictive understanding of NF?B in response to specific I?B kinase (IKK) activity profiles, and in response to the cytokine TNF and ribotoxic stress agents. However, preliminary results suggest that TLR signaling cannot be accounted for by a single kinase input into the NF?B signaling module, but that multiple regulatory mechanisms converge to regulate NF?B. Here we will undertake quantitative experimental studies in conjunction with computational modeling to produce a predictive understanding of the regulation of the key inflammatory regulator NF?B and its associated gene expression programs in response to a variety of TLR agonists. Three specific aims focus on specific mechanisms that critically modulate NF?B activity in macrophages in response to specific input signals; the fourth aim integrates these to produce a comprehensive predictive model of TLR signaling to NF?B. We will demonstrate its utility as a research tool and will provide it to the broader community.